Quantitative analysis of vesicle recycling at the calyx of Held synapse

Vesicle recycling is pivotal for maintaining reliable synaptic signaling, but its basic properties remain poorly understood. Here, we developed an approach to quantitatively analyze the kinetics of vesicle recycling with exquisite signal and temporal resolution at the calyx of Held synapse. The combination of this electrophysiological approach with electron microscopy revealed that ∼80% of vesicles (∼270,000 out of ∼330,000) in the nerve terminal are involved in recycling. Under sustained stimulation, recycled vesicles start to be reused in tens of seconds when ∼47% of the preserved vesicles in the recycling pool (RP) are depleted. The heterogeneity of vesicle recycling as well as two kinetic components of RP depletion revealed the existence of a replenishable pool of vesicles before the priming stage and led to a realistic kinetic model that assesses the size of the subpools of the RP. Thus, our study quantified the kinetics of vesicle recycling and kinetically dissected the whole vesicle pool in the calyceal terminal into the readily releasable pool (∼0.6%), the readily priming pool (∼46%), the premature pool (∼33%), and the resting pool (∼20%).Synaptic vesicle recycling ensures synaptic transmission during sustained neuronal activity (1–3). Despite its crucial role, the cycle is poorly understood. In contrast to vesicle exocytosis and endocytosis, which can be directly assayed by presynaptic capacitance measurements and postsynaptic current recordings, vesicle recycling is usually investigated by fluorescence imaging and electron microscopy (EM) with limited signal or temporal resolution (4–7). Likely owing to technical difficulties, the basic properties of vesicle recycling, such as the size of the recycling pool (RP) (3, 6, 8–11), the kinetics of vesicle recycling (6, 8–12), and how the RP supports synaptic transmission (1, 13–15) remain to be elucidated. Classically, presynaptic vesicles can be functionally divided into three populations: the readily releasable pool (RRP), the reserve pool, and the resting pool (3, 16, 17). The RRP is defined as being composed of docked and immediately releasable vesicles (17), which are usually depleted by high-frequency stimulation, prolonged presynaptic depolarization, or the application of hypertonic solution (18–21). The reserve pool functions as a reservoir and serves to maintain vesicle refilling into the RRP (2, 3). These two pools together are commonly referred to as the RP. The resting pool serves as a depot of vesicles for backup use (16, 22). However, it has been debated for a decade whether nerve terminals use the majority (∼100%, from electrophysiology) or only a small fraction (5–40%, from fluorescence imaging and EM) of vesicles in recycling, and whether the RP size undergoes dynamic changes during varied neuronal activity (6, 7, 23–28).The use of vesicles in recycling is a critical determinant of synaptic transmission (1, 13–15). However, it has never been rigorously determined how fast recently recaptured vesicles are organized to recycle and whether vesicles in the RP are homogeneously ready for use (25). Two forms of vesicle retrieval, “kiss-and-run” and full collapse, have been reported for many years. It is still ambiguous whether the rapidly recaptured vesicles in the kiss-and-run mode can be rapidly reused (29–31).Here, we addressed the above issues by developing a new approach to quantify the basic properties of vesicle recycling with unparalleled precision. Different from previous studies in cultured cell systems, the present work combined electrophysiological measurements and EM observations at the calyx of Held synapse in acute brain slices, quantitatively analyzed synaptic vesicle recycling, and kinetically dissected the recycling vesicle pool. We propose a realistic kinetic model and provide new insights into the mechanism that ensures rate-limited but sustainable synaptic transmission.     

Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β2AR

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β₂-adrenergic receptor (β₂AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.G protein-coupled receptors (GPCRs) mediate a multitude of physiological functions and are the targets for a myriad of drugs (1), many of which elicit different functional outcomes through the same receptor (2). It remains to be rationalized at the molecular level why some drugs stimulate the signaling activity of a GPCR (full or partial agonists), whereas others either repress the receptor (inverse agonists) or have no effect on the intrinsic signaling activity (neutral antagonists). Moreover, the existence of a high basal activity of some GPCRs (3) suggests that the conformational transitions leading to activation may occur spontaneously, even in the absence of ligands, which in turn raises questions about the mechanistic roles of GPCR ligands. Understanding the mechanisms and pathways of receptor activation or deactivation, and how these are linked to the binding of ligands with different chemical structures and pharmacological efficacies, will aid in design of new GPCR-targeted drugs with tailored pharmacological responses and fewer side effects. To attain these goals, new methods are required to visualize the conformational dynamics of GPCRs in the presence and absence of drugs.The β₂-adrenergic receptor (β₂AR) has been extensively investigated in crystals (4, 5), by NMR in solution (6–11), by bulk fluorescence spectroscopy in solution (12–14) and in cells (2), by single-molecule fluorescence spectroscopy (15–17), and by molecular dynamics simulations (18). Despite the availability of high-resolution crystal structures of β₂AR in inactive (4) and active (5) conformations, it remains unknown how ligands regulate transitions between the two states and why β₂AR exhibits a significant level of ligand-independent, basal signaling activity. To address these questions, we use single-molecule fluorescence spectroscopy to monitor activation-linked conformational transitions of individual receptor molecules in real time over extended time periods. Our results highlight the intrinsically dynamic character of β₂AR and provide insights into the mechanism of receptor activation and the roles of β₂AR ligands.     

Separation of on-target efficacy from adverse effects through rational design of a bitopic adenosine receptor agonist

The concepts of allosteric modulation and biased agonism are revolutionizing modern approaches to drug discovery, particularly in the field of G protein-coupled receptors (GPCRs). Both phenomena exploit topographically distinct binding sites to promote unique GPCR conformations that can lead to different patterns of cellular responsiveness. The adenosine A₁ GPCR (A₁AR) is a major therapeutic target for cardioprotection, but current agents acting on the receptor are clinically limited for this indication because of on-target bradycardia as a serious adverse effect. In the current study, we have rationally designed a novel A₁AR ligand (VCP746)—a hybrid molecule comprising adenosine linked to a positive allosteric modulator—specifically to engender biased signaling at the A₁AR. We validate that the interaction of VCP746 with the A₁AR is consistent with a bitopic mode of receptor engagement (i.e., concomitant association with orthosteric and allosteric sites) and that the compound displays biased agonism relative to prototypical A₁AR ligands. Importantly, we also show that the unique pharmacology of VCP746 is (patho)physiologically relevant, because the compound protects against ischemic insult in native A₁AR-expressing cardiomyoblasts and cardiomyocytes but does not affect rat atrial heart rate. Thus, this study provides proof of concept that bitopic ligands can be designed as biased agonists to promote on-target efficacy without on-target side effects.G protein-coupled receptors (GPCRs) are the largest family of cell surface proteins and tractable drug targets (1, 2). Unfortunately, there remains a high attrition rate associated with traditional GPCR-based drug discovery that, in part, reflects an emphasis on the endogenous agonist binding (orthosteric) site as the predominant means of achieving selective GPCR drug targeting (3). Over the last decade, substantial breakthroughs have occurred in the exploitation of topographically distinct GPCR allosteric sites as a means for attaining greater selectivity, especially in those instances where there is high sequence similarity in the orthosteric site across GPCR subtypes (4–6). However, there are increasing examples where both the therapeutic effect and adverse effects are mediated by the same GPCR target (7). In these situations, the desired selectivity needs to be attained at the level of the intracellular signaling pathways linked to a given receptor subtype.GPCRs are highly dynamic proteins, fluctuating between different conformations; these conformations can be linked to different cellular outcomes (8). Thus, chemically distinct ligands, interacting with either orthosteric or allosteric sites, have the potential to stabilize different interaction networks within a GPCR to promote a subset of signaling pathways linked to the receptor at the expense of others. This phenomenon has been termed biased agonism (7, 9, 10). The overall promise of biased agonism is the ability to design GPCR ligands that selectively engage therapeutically relevant signaling pathways while sparing pathways that contribute to undesirable side effects mediated by the same target.The adenosine receptor (AR) family is an important class of physiologically and therapeutically relevant GPCRs that can benefit substantially from more selective drug targeting. Although all four AR subtypes are expressed in the mammalian heart (11, 12), the well-known protective effects of adenosine in this tissue are predominantly mediated by the adenosine A₁ receptor (A₁AR) subtype, especially under conditions of ischemia and reperfusion injury (13–17). Unfortunately, the transition of A₁AR agonists into the clinic has been severely hindered because of high doses causing on-target bradycardia, atrioventricular block, and hypotension (13, 18). As a consequence, clinical trials of AR agonists have had limited success because of the suboptimal dose of agonist that can be used (19–22). It is possible that this problem may be overcome through the exploitation of biased agonism at the A₁AR.Although no study has identified biased orthosteric A₁AR ligands, we recently showed that the 2-amino-3-benzoylthiophene allosteric modulator (VCP171) could promote biased signaling in the activity of the prototypical orthosteric agonist, R(-)N6-(2-phenylisopropyl) adenosine (R-PIA) (23). Thus, we hypothesized that the rational design of a bitopic ligand (i.e., a class of hybrid molecule containing both orthosteric and allosteric pharmacophores) (24–26) may be able to achieve high efficacy and biased agonism at the A₁AR in a single molecule. Herein, we report proof of concept that it is possible to use this approach as a means to dissociate on-target efficacy from on-target side effects.     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.     

OPG/RANKL/RANK axis is a critical inflammatory signaling system in ischemic brain in mice

Osteoprotegerin (OPG) is a soluble secreted protein and a decoy receptor, which inhibits a receptor activator of nuclear factor κB (NF-κB) ligand (RANKL)/the receptor activator of NF-κB (RANK) signaling. Recent clinical studies have shown that a high-serum-OPG level is associated with unfavorable outcome in ischemic stroke, but it is unclear whether OPG is a culprit or an innocent bystander. Here we demonstrate that enhanced RANKL/RANK signaling in OPG^−/− mice or recombinant RANKL-treated mice contributed to the reduction of infarct volume and brain edema via reduced postischemic inflammation. On the contrary, infarct volume was increased by reduced RANKL/RANK signaling in OPG^−/− mice and WT mice treated with anti-RANKL neutralizing antibody. OPG, RANKL, and RANK mRNA were increased in the acute stage and were expressed in activated microglia and macrophages. Although enhanced RANKL/RANK signaling had no effects in glutamate, CoCl₂, or H₂O₂-stimulated neuronal culture, enhanced RANKL/RANK signaling showed neuroprotective effects with reduced expression in inflammatory cytokines in LPS-stimulated neuron-glia mixed culture, suggesting that RANKL/RANK signaling can attenuate inflammation through a Toll-like receptor signaling pathway in microglia. Our findings propose that increased OPG could be a causal factor of reducing RANKL/RANK signaling and increasing postischemic inflammation. Thus, the OPG/RANKL/RANK axis plays critical roles in controlling inflammation in ischemic brains.An elevated serum osteoprotegerin (OPG) level has been reported to be associated with the severity (1, 2), subtype (2), poor functional outcome (1), and long-term mortality of ischemic stroke (2, 3). However, it is still unclear why a high-serum-OPG level could result in a poor prognosis in ischemic stroke.OPG is a soluble secreted protein that lacks transmembrane and cytoplasmic domains. It binds to a receptor activator of nuclear factor-кB ligand (RANKL) (4, 5), whose receptor is the receptor activator of NF-κB (RANK), and inhibits RANKL/RANK signaling. The OPG/RANKL/RANK system in bone (6) and vasculature (7) is well known to work on bone metabolism (8) and vascular calcification (7). In addition, immune cells (6, 9–12) express these molecules, and this system is believed to be associated with the regulation of inflammatory and immune responses (13, 14). RANKL is expressed in CD4⁺ T cells (6) and macrophages (9, 10), whereas OPG is expressed in mature B cells (6) and macrophages (11, 12). RANK is expressed in macrophage and dendritic cells (6). One of the functions of RANKL/RANK signaling in the immune system is to control the thymocyte-mediated medulla formation and the formation of self-tolerance in T cells (14), as well as the number of regulatory T cells (Treg) (15). Additionally, RANKL directly contributes to the regulation of proinflammatory cytokine production in macrophages (13, 16).Despite these characteristics of the OPG/RANKL/RANK system, its action on inflammation in central nervous system diseases has yet to be studied. Ischemic stroke is a typical acute inflammatory disease, and postischemic inflammation affects the outcome, i.e., the infarct volume (17). These inflammatory cytokines are produced mainly from microglia in the early phase and from mixed microglia/macrophage (M/M) in the delayed phase, i.e., 12–24 h after ischemia, whereas neutrophilic granulocytes are not responsible for the production of those inflammatory cytokines (17). Because OPG/RANKL/RANK is expressed in macrophages (6, 9–12) and affects inflammatory responses (13), we hypothesized that one of the mechanisms of poor outcome in high-level OPG might reflect the modulation of such postischemic inflammations by the OPG/RANKL/RANK signaling system.Interestingly, OPG, RANKL, and RANK have also been reported to be expressed in normal brain in rodents, although the distribution of their expression is controversial. Early reports showed that RANKL mRNA was expressed in neurons in the cerebral cortex (18) and that RANK (19) and OPG (5) mRNA was expressed in normal brain. However, a recent report revealed that the RANK protein was specifically expressed in neurons and astrocytes in the preoptic area and the medial septal nucleus, whereas RANKL mRNA was expressed in the lateral septal nucleus (20). In normal brain, RANKL/RANK signaling was reported to be associated with fever and body temperature control (20). Because body temperature is associated with the outcome in ischemic stroke, we also hypothesized that the modulation of body temperature by the OPG/RANKL/RANK system might be another mechanism behind a poor outcome in ischemic stroke. To clarify these hypotheses, we examined the action of the OPG/RANKL/RANK signaling system in a transient middle cerebral artery occlusion (MCAo) model using OPG^−/− mice and treated RANK fragment crystalizable (Fc) chimera to inhibit the function of RANKL or recombinant RANKL.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Nicotinic and muscarinic agonists and acetylcholinesterase inhibitors stimulate a common pathway to enhance GluN2B-NMDAR responses

Nicotinic and muscarinic ACh receptor agonists and acetylcholinesterase inhibitors (AChEIs) can enhance cognitive function. However, it is unknown whether a common signaling pathway is involved in the effect. Here, we show that in vivo administration of nicotine, AChEIs, and an m1 muscarinic (m1) agonist increase glutamate receptor, ionotropic, N-methyl D-aspartate 2B (GluN2B)-containing NMDA receptor (NR2B-NMDAR) responses, a necessary component in memory formation, in hippocampal CA1 pyramidal cells, and that coadministration of the m1 antagonist pirenzepine prevents the effect of cholinergic drugs. These observations suggest that the effect of nicotine is secondary to increased release of ACh via the activation of nicotinic ACh receptors (nAChRs) and involves m1 receptor activation through ACh. In vitro activation of m1 receptors causes the selective enhancement of NR2B-NMDAR responses in CA1 pyramidal cells, and in vivo exposure to cholinergic drugs occludes the in vitro effect. Furthermore, in vivo exposure to cholinergic drugs suppresses the potentiating effect of Src on NMDAR responses in vitro. These results suggest that exposure to cholinergic drugs maximally stimulates the m1/guanine nucleotide-binding protein subunit alpha q/PKC/proline-rich tyrosine kinase 2/Src signaling pathway for the potentiation of NMDAR responses in vivo, occluding the in vitro effects of m1 activation and Src. Thus, our results indicate not only that nAChRs, ACh, and m1 receptors are on the same pathway involving Src signaling but also that NR2B-NMDARs are a point of convergence of cholinergic and glutamatergic pathways involved in learning and memory.Nicotinic and muscarinic agonists can produce cognitive enhancement (1, 2). Acetylcholinesterase inhibitors (AChEIs) also cause cognitive enhancement by increasing ACh levels (3, 4). However, it is largely unknown whether the effect of ACh is mediated by nicotinic ACh receptors (nAChRs), muscarinic receptors, or both. Studies involving cholinergic lesions and local administration of cholinergic antagonists indicate that both nAChRs and muscarinic receptors located in the hippocampus are of particular importance for learning and memory processes (5–8). However, the mechanisms by which these receptors mediate cognitive enhancement largely remain to be elucidated.Synaptic plasticity is thought to be a critical component underlying learning and memory (9, 10), and the NMDA receptor (NMDAR) is a key component of synaptic plasticity (9, 11). Thus, studies of the modulation of NMDAR responses and long-term potentiation (LTP) induction by cholinergic drugs (12–20) help elucidate the mechanisms of cholinergic facilitation of learning and memory. In vitro acute nicotine can potentiate NMDAR-mediated responses in CA1 pyramidal cells in hippocampal slices via at least two different mechanisms (16, 18). One of these mechanisms is absent after a selective cholinergic lesion (21) and is paradoxically blocked by the muscarinic antagonist atropine (18), suggesting not only a critical role of nicotine-induced ACh release but also the involvement of muscarinic receptor activation in the effect of nicotine. This pathway appears to be stimulated by systemic nicotine administration in rats and most likely involves Src signaling (18, 19), which is known to be initiated via acute activation of m1 muscarinic (m1) receptors in CA1 pyramidal cells (22). An implication of these observations is that there is a common signaling pathway stimulated by cognitive-enhancing cholinergic drugs, leading to the enhancement of NMDAR-mediated responses in CA1 pyramidal cells. Thus, in this study, we investigated the link between nicotine and NMDARs in rats by administrating drugs that target different cholinergic proteins.     

α2A adrenergic receptor promotes amyloidogenesis through disrupting APP-SorLA interaction

Accumulation of amyloid β (Aβ) peptides in the brain is the key pathogenic factor driving Alzheimer’s disease (AD). Endocytic sorting of amyloid precursor protein (APP) mediated by the vacuolar protein sorting (Vps10) family of receptors plays a decisive role in controlling the outcome of APP proteolytic processing and Aβ generation. Here we report for the first time to our knowledge that this process is regulated by a G protein-coupled receptor, the α_2A adrenergic receptor (α_2AAR). Genetic deficiency of the α_2AAR significantly reduces, whereas stimulation of this receptor enhances, Aβ generation and AD-related pathology. Activation of α_2AAR signaling disrupts APP interaction with a Vps10 family receptor, sorting-related receptor with A repeat (SorLA), in cells and in the mouse brain. As a consequence, activation of α_2AAR reduces Golgi localization of APP and concurrently promotes APP distribution in endosomes and cleavage by β secretase. The α_2AAR is a key component of the brain noradrenergic system. Profound noradrenergic dysfunction occurs consistently in patients at the early stages of AD. α_2AAR-promoted Aβ generation provides a novel mechanism underlying the connection between noradrenergic dysfunction and AD. Our study also suggests α_2AAR as a previously unappreciated therapeutic target for AD. Significantly, pharmacological blockade of the α_2AAR by a clinically used antagonist reduces AD-related pathology and ameliorates cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α₂AR antagonists would be an effective therapeutic strategy for AD.Excess amyloid β (Aβ) peptides in the brain are a neuropathological hallmark of Alzheimer’s disease (AD) and are generally accepted as the key pathogenic factor of the disease (1). Aβ is generated by two sequential cleavages of amyloid precursor protein (APP) by β and γ secretase, whereas cleavage by α secretase within the Aβ domain precludes Aβ generation (2, 3). APP and the secretases undergo endocytic sorting into various organelles, such as the trans-Golgi network, the plasma membrane, and endosomes (2–6). The initial step of APP processing by α versus β secretase preferentially occurs in distinct compartments of the cell. Although α secretase-mediated cleavage of APP occurs on the plasma membrane, β secretase primarily interacts with and cleaves APP in endosomes (2–6). Therefore, endocytic sorting of APP into different membranous compartments, causing it to coreside or avoid a particular secretase, plays a decisive role in APP proteolytic processing. Consistent with this notion, abnormalities of the endocytic pathway have been found to precede Aβ deposition in late-onset AD (7).Retrograde sorting of APP from endosomes to trans-Golgi network mediated by the vacuolar protein sorting-10 (Vps10) family proteins and the retromer complex represents a critical mechanism to prevent amyloidogenic processing of APP (8–10) and has recently emerged as a potential target for therapeutic intervention (11). In particular, the sorting-related receptor with A repeat (SorLA) directs retrograde transport of APP to trans-Golgi network by binding to both APP and the retromer complex (12, 13) and retains APP in the Golgi (14), thus preventing its proteolytic processing. A connection between SorLA and AD was first revealed in patients with late-onset AD, in whom the levels of SorLA at the steady state are markedly reduced (15). Further human genetic studies identified variations of SORL1 (the gene encoding SorLA) resulting in a lower level of expression that are associated with late-onset sporadic AD (12, 16, 17). Moreover, nonsense and missense mutations of SORL1 cause autosomal dominant early-onset AD (18), supporting an etiological role of SorLA in AD. The function of SorLA in inhibiting Aβ production is confirmed by mouse genetic studies showing that loss of SorLA significantly increases Aβ levels in the brain (14) and enhances AD-related early pathology (19). Despite the importance of SorLA-dependent APP sorting in controlling Aβ metabolism and AD pathogenesis, how this process may be targeted by extracellular stimuli, such as neurotransmitters and hormones, to modulate amyloidogenesis remains largely unstudied.The α_2A adrenergic receptor (AR) belongs to the G protein-coupled receptor (GPCR) superfamily and is a crucial component of the brain noradrenergic (NA) system, controlling both NA input to the cerebral cortex and the resulting response in this brain region (20). Profound dysfunction of the NA system consistently occurs at the early stage of AD (21), raising the possibility of involvement of the α_2AAR in AD pathogenesis. Here we report for the first time to our knowledge that α_2AAR signaling regulates SorLA-dependent APP sorting and promotes amyloidogenic processing of APP by beta-site amyloid precursor protein cleaving enzyme (BACE) 1. The initial cleavage of APP by BACE1 is the rate-limiting factor of Aβ generation (22, 23). Furthermore, blockade of α_2AAR by a clinical antagonist reduces AD-related pathology and rescues cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α₂AR antagonists would be a novel effective strategy for AD treatment.     

From the Cover: PNAS Plus: Timing of androgen receptor disruption and estrogen exposure underlies a spectrum of congenital penile anomalies

Congenital penile anomalies (CPAs) are among the most common human birth defects. Reports of CPAs, which include hypospadias, chordee, micropenis, and ambiguous genitalia, have risen sharply in recent decades, but the causes of these malformations are rarely identified. Both genetic anomalies and environmental factors, such as antiandrogenic and estrogenic endocrine disrupting chemicals (EDCs), are suspected to cause CPAs; however, little is known about the temporal window(s) of sensitivity to EDCs, or the tissue-specific roles and downstream targets of the androgen receptor (AR) in external genitalia. Here, we show that the full spectrum of CPAs can be produced by disrupting AR at different developmental stages and in specific cell types in the mouse genital tubercle. Inactivation of AR during a narrow window of prenatal development results in hypospadias and chordee, whereas earlier disruptions cause ambiguous genitalia and later disruptions cause micropenis. The neonatal phase of penile development is controlled by the balance of AR to estrogen receptor α (ERα) activity; either inhibition of androgen or augmentation of estrogen signaling can induce micropenis. AR and ERα have opposite effects on cell division, apoptosis, and regulation of Hedgehog, fibroblast growth factor, bone morphogenetic protein, and Wnt signaling in the genital tubercle. We identify Indian hedgehog (Ihh) as a novel downstream target of AR in external genitalia and show that conditional deletion of Ihh inhibits penile masculinization. These studies reveal previously unidentified cellular and molecular mechanisms by which antiandrogenic and estrogenic signals induce penile malformations and demonstrate that the timing of endocrine disruption can determine the type of CPA.Congenital penile anomalies (CPAs) encompass a spectrum of malformations of the penis. Analysis of the Nationwide Inpatient Sample, the largest inpatient database in the United States, identified CPA in 7.8/1,000 newborns and showed that the frequency of CPA has increased over the past 40 y (1–3). The most common CPA is hypospadias (68.3%), followed by chordee (8.6%) and hypospadias plus chordee (5%), and 14% are reported as unspecified penile anomalies (2). The range of structural defects included in the CPA classification suggests that a single developmental mechanism is unlikely to account for the full spectrum of malformations. Furthermore, the rate at which reports of CPAs have increased in recent decades cannot be explained by genetics alone. There is increasing evidence that environmental factors, particularly exposures to environmental endocrine disrupting chemicals (EDCs), may play a causal role in these developmental defects (4); however, little is known about the interactions between EDCs and the gene networks that control external genital development, the temporal windows of sensitivity to EDC exposure, the endogenous role(s) of estrogen in penile development (5–7), or the relationship between androgen and estrogen signaling in normal genital development. At present, there are no mouse models for human-like CPAs, such as midshaft hypospadias (with or without chordee) or micropenis.Hypospadias is a urethral tube defect in which the urethra opens ectopically on the ventral side of the penis, between the glans and the perineum. The severity of hypospadias can range from a slightly offset urethral meatus to complete failure of urethral tube formation, which can result in ambiguous genitalia. Epispadias is a less common urethral tube defect, in which the urethra opens on the dorsal side of the penis. Both malformations can be associated with chordee, an abnormal bending of the penis, which may involve soft-tissue tethering. Hypospadias and epispadias can occur in both sexes although detection in females is challenging (8, 9). Micropenis refers to an abnormally small but normally structured penis with a stretched penile length of >2.5 SDs below the mean human penis size for the same age individual (10, 11). Micropenis is often associated with both functional (related to sex and voiding) and psychological problems, and patients with micropenis can suffer from penile dysmorphic disorders (12). Analyses of mouse mutants have implicated a number of developmental control genes in hypospadias (reviewed in ref. 13), and, although association studies of affected patients have identified promising candidate mutations and copy number variants (14–16), the causes of hypospadias in humans remain largely unknown (17).Androgens and estrogen are steroid hormones that play critical roles in sexually dimorphic genital development (18). Masculinization of male external genitalia is determined mainly by androgen signaling. Mice with mutations in the androgen receptor (AR) or 5α-reductase, which converts testosterone to dihydrotestosterone, develop feminized external genitalia (19, 20). In humans, mutations in these genes underlie androgen insensitivity syndrome, in which genetic males fail to respond to androgen signaling and consequently develop feminized genitalia and secondary sex characters (21). Prenatal exposure to antiandrogenic chemicals can feminize the external genitalia and induce hypospadias in male rodents (22–25).It has long been thought that female external genitalia develop due to the low levels of androgen signaling activity; however, recent studies of mouse estrogen receptor α (ERα) mutants revealed virilization of the clitoris, demonstrating that estrogen signaling via ERα is required for normal development of female external genitalia (26). Deletion of ERα in mice affects only female external genitalia development (26) although mutations in ERα have been identified in human males with genital and reproductive abnormalities (27). Furthermore, exposure of neonatal rats to estrogen results in reduced penis size and weight, an effect mediated by ERα (26, 28, 29). Deletion of ERα is sufficient to rescue diethylstilbestrol (DES)-induced feminization of the penis (29), suggesting that ERα plays a crucial role in mediating estrogen-driven penile anomalies.We combined mouse genetics with pharmacology to dissect the temporal and tissue-specific roles of androgen and estrogen signaling in penile development, and we show that disruption of AR and/or activation of ERα at discrete time periods can produce the complete spectrum of congenital penile anomalies found in humans. Here, we show that normal development of male external genitalia in mice involves two phases of AR and ERα activity; an early prenatal phase regulates formation of a closed urethral tube and development of the surrounding stromal tissue and prepuce, and a later neonatal phase controls proliferation of the glans penis mesenchyme. Our results demonstrate that modulation of androgen and estrogen signaling at discrete developmental time points can account for the entire spectrum of CPAs.     

FCRL5 exerts binary and compartment-specific influence on innate-like B-cell receptor signaling

Innate-like splenic marginal zone (MZ) and peritoneal cavity B1 B lymphocytes share critical responsibilities in humoral responses but have divergent B-cell receptor (BCR) signaling features. A discrete marker of these subsets with tyrosine-based dual regulatory potential termed “Fc receptor-like 5” (FCRL5) was investigated to explore this discrepancy. Although FCRL5 repressed the robust BCR activity that is characteristic of MZ B cells, it had no influence on antigen receptor stimulation that is blunted in peritoneal cavity-derived B1 B cells. The molecular basis for the receptor’s inhibitory function derived from recruitment of the Src homology-2 domain-containing tyrosine phosphatase 1 (SHP-1) to a cytoplasmic immunoreceptor tyrosine-based inhibitory motif. Surprisingly, mutagenesis of this docking site unearthed coactivation properties for FCRL5 that were orchestrated by independent association of the Lyn Src-family kinase with an intracellular immunoreceptor tyrosine-based activation motif-like sequence. FCRL5’s unique binary regulation directly correlated with SHP-1 and Lyn activity, which, like BCR function, differed between MZ and B1 B cells. These findings collectively imply a specialized counterregulatory role for FCRL molecules at the intersection of innate and adaptive immunity.Innate-like B-lineage cells positioned at strategic microanatomical sites provide the first line of effector defense that bridges host protection until adaptive mechanisms emerge (1). The lymphocytes charged with these responsibilities include splenic marginal zone (MZ) B cells harbored in a location optimal for filtering blood-borne antigens and B1-lineage cells that guard the peritoneal (PEC) and pleural body cavities (2–4). Their capacity for broad neutralization is associated with evolutionarily conserved Ig repertoires, distinct sensitivity to T-cell–independent stimuli, and rapid or spontaneous production of ‘‘natural,’’ polyreactive antibodies (5, 6). These features distinguish MZ and B1 B cells from their more abundant B2-lineage counterparts that recirculate and participate in T-cell–dependent responses.MZ and B1 B-cell development is strongly influenced by B-cell receptor (BCR) specificity in concert with the composite array of surface and intracellular regulatory proteins that help balance antigenic responses. Mutations in cluster of differentiation 45 (CD45), Bruton''s tyrosine kinase (BTK), or phospholipase C gamma 2 (PLCγ2) that dampen BCR signaling favor MZ development and a loss of follicular (FO) B cells (7). However, defects in negative regulatory components, such as Lyn, Src homology-2 (SH2) domain-containing tyrosine phosphatase 1 (SHP-1), or CD22, lead to a loss of MZ B cells, a relative expansion of the B1 compartment, and increased susceptibility to autoimmunity (8). Although many other trophic, migratory, and retention factors instruct their development and positioning, these signals must be integrated in the context of BCR signaling which primarily drives B-cell fate and survival (7, 9–11). Correspondingly, BCR triggering differs markedly between these subpopulations. MZ B cells exhibit more robust whole-cell protein tyrosine phosphorylation, calcium mobilization, and PLCγ2 and spleen tyrosine kinase (Syk) activation than FO B cells but also are more sensitive to apoptosis (12, 13). In contrast, B1 B cells have blunted calcium mobilization, NF-κB activation, and proliferation but also may possess relatively higher rates of apoptosis than PEC B2 cells (14–16). Notably, these properties do not differ according to CD5 expression, because both the B1a (CD5⁺) and B1b (CD5⁻) B-cell subsets respond similarly to BCR ligation (17). Although they differ from anergic cells, it remains unclear whether the unique biology of B1 B cells is secondary to chronic antigenic exposure, suppressive signals present in the coelomic cavity microenvironment, or other causes (4, 14).An evolutionarily conserved gene family related to the Fc receptors (FCR) for IgG and IgE, termed “FCR-like” (FCRL), is preferentially expressed by B cells and encodes transmembrane proteins with tyrosine-based immunoregulatory motifs (18). Although Ig binding has been detected recently for two human members (19), antibodies do not appear to associate with other FCRL proteins. Intriguingly, two other representatives have been found to interact with MHC-like molecules (20, 21). Because of the growing clinical relevance of FCRLs in infectious diseases, autoimmunity, malignancies, and immunodeficiencies, several groups have investigated FCRL signaling function (22–25). In humans, FCRL1 has two immunoreceptor tyrosine-based activation motif (ITAM)-like sequences and enhances BCR-induced calcium mobilization and cellular proliferation (26). In contrast, FCRL2–5, which feature one or more consensus immunoreceptor tyrosine-based inhibition motifs (ITIM) as well as ITAM-like sequences, all inhibit BCR activation via recruitment of the SH2 domain-containing SHP-1 and/or SHP-2 phosphatases (27–30). We have shown previously that the FCRL5 mouse ortholog discretely marks innate-like B cells and possesses an ITIM as well as an ITAM-like sequence that differs from the canonical motif (D/EX_2–3YXXL/IX_6–8YXXL/I), with a glutamic acid residue rather than an aliphatic residue at the second Y+3 position (31). Although FCRL5 inhibits BCR-mediated calcium mobilization in MZ B cells, the molecular basis for this activity and its function in B1 B cells remains unclear. Furthermore, the conservation of both activating and inhibitory sequences in FCRL5 and other FCRLs suggests they have dual signaling properties, but definitive functional evidence for this bifunctionality is lacking. Because of its distinct distribution and regulatory potential, we investigated the biological role of FCRL5 in these specialized B lymphocytes that have recognized differences in their adaptive signaling capacity.Here we report that FCRL5 has dual modulatory and compartmental subset-specific effects on antigen receptor signaling. Upon association with the BCR, the ITAM-like and ITIM sequences in FCRL5 are tyrosine phosphorylated and recruit the Lyn Src-family kinase (SFK) and SHP-1 protein tyrosine phosphatase. The nonredundant contributions of these elements to FCRL5’s unique counterregulatory function further revealed that differences in adaptive signaling in MZ and B1 B cells correlate directly with their intrinsic SHP-1 activity.